Abscisic acid is a central stress hormone in Arabidopsis which mediates rapid Ca2+-induced cellular responses, down regulates cell proliferation and inhibits growth. Early signal transduction mechanisms that down regulate cell proliferation are of central importance for controlling mitogenesis and their mis-regulation is linked to many human diseases. Abscisic acid (ABA)-triggered Ca2+ signaling will be analyzed in Arabidopsis guard cells, which provide a powerful system for quantitative and time- resolved dissection of mechanisms mediating specificity in Ca2+ signaling. How the universal second messenger Ca2+ mediates specific responses in eukaryotic cells, is a central question in cell signaling research. Data from several lines of our investigations point to a novel hypothesis for specificity in Ca2+ signal transduction, in which the physiological stimulus, ABA, "primes" (de-inactivates) specific Ca2+ sensors, switching them from an inactivated Ca2+-insensitive state to a Ca2+-sensitive "primed" state, thus tightly controlling Ca2+ responsiveness. Calcium sensitivity priming could provide a key mechanism contributing to specificity in eukaryotic Ca2+ signal transduction. We have recently discovered major signaling mechanisms and genes that form this ABA-regulated-cytosolic Ca2+ signaling module, including Ca2+ channel genes, Ca2+ sensors (CDPKs) and major downstream targets of Ca2+: a long-sought membrane protein, RCD3, required for Ca2+-activated anion channel activity, which is essential for the ABA-Ca2+ response, and an ABC transmembrane channel regulator (AtMRP5). We will investigate the mechanisms mediating Ca2+ signaling specificity by pursuing the following specific aims: (1) We will dissect the mechanisms by which ABA activates newly identified Ca2+ channel genes and determine their functions in generating guard cell Ca2+ oscillations. (2) Characterize possible Ca2+ specificity signaling mechanisms by analyzing ABA-dependent cellular localization and modulation of the CDPK Ca2+ sensors and associated proteins. (3) We will systematically dissect the protein- protein interaction network through which these newly isolated major Ca2+ signaling mechanisms interact, thus enabling ABA-directed specificity in Ca2+ signaling. (4) We have developed a powerful chemical genetics screen for ABA/Ca2+ signaling, using a compound that inhibits Ca2+-induced stomatal closing, and that can address genetic redundancy and signaling robustness. This has enabled us to isolate new early ABA signaling mutants that will be functionally characterized within the ABA-Ca2+ signaling network. These studies should advance a mechanistic understanding of specificity in Ca2+ signaling, which is fundamental to numerous cell signaling processes, and will reveal novel mechanisms by which a newly recognized early signaling pathway controls ABA signal transduction. Project Narrative How the universal second messenger Ca2+ mediates specific responses in eukaryotic cells, is a key question in cell signaling research. This research will reveal major mechanisms by which the early abscisic acid-directed calcium signal transduction pathway mediates stress hormone signaling. These studies should substantially advance an understanding of the mechanisms mediating specificity in Ca2+ signaling in a highly developed model cell system, a basic process that is fundamental to numerous cell signaling pathways and is mis-regulated in human diseases.